Synergistic antimicrobial effects of bacteriocins and essential oils are an effective way to address food safety issues caused by foodborne pathogens, but the related synergistic antibacterial mechanisms are still unclear. This study investigated the synergistic antibacterial mechanisms of enterocin Gr17 and cinnamaldehyde against Listeria monocytogenes from the perspectives of morphological structure, cell wall, cell membrane and energy metabolism. Time-killing experiments showed that enterocin Gr17 and cinnamaldehyde synergistically inhibited the growth of Listeria monocytogenes in a time-dependent manner, with a reduction of bacterial counts by 6.22lg CFU/mL for 24h. Scanning electron microscopy and transmission electron microscopy analysis revealed that enterocin Gr17 and cinnamaldehyde synergistically disrupted the morphology and internal tissue of cells, resulting in the disintegration of cell structure. Alkaline phosphatase activity and Zeta potential analysis indicated that enterocin Gr17 and cinnamaldehyde synergistically disrupted the cell wall integrity of Listeria monocytogenes via disrupting its surface potential and targeting cell wall constituents. The leakage of K⁺ and adenosine triphosphate and the massive release of nucleic acids, proteins, and lactate dehydrogenase indicated that enterocin Gr17 and cinnamaldehyde synergistically formed non-selective pores in the cell membrane of Listeria monocytogenes, disrupted membrane permeability, further inhibited energy metabolism, and ultimately caused irreversible structural damage to the membrane integrity and cell death. Notably, enterocin Gr17 mainly targeted and destroyed the cell wall, while cinnamaldehyde primarily acted on the cell membrane. Their synergistic interaction exacerbated the loss of structural integrity in both the cell wall and membrane, culminating in compromised energy metabolism. This study provided a theoretical reference and scientific basis for understanding the synergistic antimicrobial mechanism of natural antimicrobial agents.